JP7370909B2 - Method for preparing hyaluronic acid, method for detecting hyaluronic acid, and kits thereof - Google Patents

Description

The present invention relates to a method and kit for preparing hyaluronic acid. The present invention also relates to a method and kit for detecting hyaluronic acid. More specifically, the present invention relates to a method for preparing hyaluronic acid derived from skin cells, a method for detecting hyaluronic acid, and a kit thereof.

Hyaluronic acid is widely present in the connective tissues of animals, such as the vitreous body of the eye, the crest of chickens, the umbilical cord, the skin, and the joint fluid. The skin contains the largest amount of hyaluronic acid in the body, and plays an important role as an extracellular matrix. It is also known that hyaluronic acid in the skin decreases with age, and is thought to be a contributing factor to skin aging phenomena such as rough skin, decreased skin elasticity and firmness, wrinkles, and sagging.

Therefore, various methods have been proposed to replenish the decreased hyaluronic acid. Examples include a method of directly ingesting hyaluronic acid and a method of ingesting foods and drinks that promote the production of hyaluronic acid. Although all of these methods have been confirmed to be effective in experiments using cultured cells, their effects have not been directly demonstrated in humans. The reason for this is that when trying to detect hyaluronic acid from the skin, a method for non-invasively and directly detecting hyaluronic acid has not been established. Therefore, a common method for confirming the effect on the human body is to measure the skin moisture content using impedance or the like and indirectly estimate the increase or decrease in hyaluronic acid (see Patent Document 1).

However, the moisture content of the skin is not necessarily determined by an increase or decrease in the amount of hyaluronic acid, but is determined by the interaction with various factors. Therefore, it is unclear whether the amount of hyaluronic acid is really changing based on impedance, etc., and there is still the problem of having to rely on invasive methods to obtain accurate data.

Patent No. 3772104

The present invention has been made in view of the above problems. That is, an object of the present invention is to provide a method and a kit that can non-invasively and directly detect and measure hyaluronic acid.

This time, the applicants conducted intensive research to find a method to detect hyaluronic acid non-invasively and directly. Then, they newly discovered that hyaluronic acid can be detected from sebum, which was previously thought not to contain hyaluronic acid, leading to the completion of the present invention.

In order to solve the above problems, the present invention provides a method for preparing hyaluronic acid derived from skin cells of a subject, which includes separating hyaluronic acid from skin surface lipids collected from the subject. The present invention also provides a method for detecting hyaluronic acid derived from skin cells of a subject, which includes a collection step of collecting lipids on the skin surface of the subject, and treating the collected lipids on the skin surface with a surfactant. The present invention provides a method for detecting hyaluronic acid derived from skin cells of a subject, which comprises a treatment step of treating hyaluronic acid with a surfactant, and a detection step of detecting hyaluronic acid treated with a surfactant. Further provided are kits for preparing and detecting hyaluronic acid derived from skin cells of a subject, including a tool for collecting lipids on the surface of the subject's skin.

According to the present invention, hyaluronic acid can be detected non-invasively and directly.

It is a graph showing the amount of hyaluronic acid contained in sebum collected from a subject. It is a graph showing the difference in the amount of hyaluronic acid extracted from sebum due to the difference in the extraction solvent.

(Preparation method of hyaluronic acid)
In one aspect, the invention provides a method for preparing hyaluronic acid derived from skin cells of a subject. A method for preparing hyaluronic acid derived from skin cells of a subject according to the present invention includes separating hyaluronic acid from skin surface lipids collected from the subject.

The subject in the method of the present invention may be any living organism that has sebum on its skin. Examples of subjects include humans and non-human mammals.

"Non-invasive" as used in the present invention means that it does not injure or cause pain to living organisms, that is, it does not impose a burden on the body.

The "skin surface lipid" as used in the present invention refers to a fat-soluble fraction present on the surface of the skin, and in this specification mainly refers to sebum. Sebum is an emulsion-like liquid secreted by the skin cells of the sebaceous glands, and is spread over the skin surface in a thin film. It is known that many of the components of sebum are composed of triglycerides, wax esters, squalene, cholesterol, etc.

The term "skin" used in the present invention refers to a region containing tissues such as the epidermis, dermis, hair follicles, sweat glands, sebaceous glands, and other glands.

Sebum collected from a subject contains hyaluronic acid produced in the subject's skin cells, preferably hyaluronic acid produced in any of the subject's epidermis, sebaceous glands, hair follicles, and sweat glands.

The method of the invention may further include collecting sebum from the subject. Examples of the skin parts from which sebum is collected include the skin of any part of the body such as the head, face, neck, trunk, limbs, and the like. It may also be skin with diseases such as atopic dermatitis, acne, inflammation, and tumors, or skin with wounds.

Any means used to collect or remove sebum from the skin can be used to collect sebum from the subject's skin. Preferably, a sebum-absorbing material, a sebum-adhesive material, or an instrument for scraping sebum from the skin, which will be described later, can be used. The sebum-absorbing material or sebum-adhesive material is not particularly limited as long as it has an affinity for sebum; for example, a method of absorbing sebum into a sheet-like material such as oil-blotting paper or oil-blotting film, or a glass plate. , a method of adhering sebum to a tape or the like, and a method of collecting sebum by scraping it off with a spatula, scraper, etc. In order to improve the adsorption of sebum, a sebum-absorbing material pre-impregnated with a highly fat-soluble solvent may be used.

(surfactant)
In order to separate hyaluronic acid from collected sebum, it is essential to treat it with a surfactant. The surfactant that can be used to extract hyaluronic acid can be selected from the group consisting of anionic, zwitterionic and nonionic surfactants.

The anionic surfactant is not particularly limited, and examples thereof include soaps having 10 to 22 carbon atoms, alkylbenzene sulfonates having 14 to 24 carbon atoms, higher alcohol sulfate salts having 10 to 22 carbon atoms, and alkyl groups having 10 carbon atoms. ~22 polyoxyethylene alkyl ether sulfates with a repeating number of oxyethylene units in the polyoxyethylene group of 1 to 10, α-sulfo fatty acid esters with 10 to 22 carbon atoms, α-olefin sulfones with 8 to 18 carbon atoms Examples include acid salts, alkanesulfonates having 10 to 22 carbon atoms, monoalkyl phosphate ester salts having 10 to 22 carbon atoms, cholates such as sodium deoxycholate, and sodium dodecyl sulfate (SDS).

The zwitterionic surfactant is not particularly limited and includes, for example, alkylamino fatty acid salts, alkyl betaines, alkyl amine oxides, myristyl sulfobetaines, and the like.

There are no particular limitations on the nonionic surfactants, including polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, alkyl glucoside, polyoxyethylene fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, and polyoxyethylene sorbitan fatty acid ester. , fatty acid alkanolamide, polyoxyethylene-polyoxypropylene block copolymer, octylphenol ethoxylate (trade name: Triton X-100), and the like.

(Applications of adjusted hyaluronic acid)
Hyaluronic acid derived from a subject's skin cells prepared by the method of the present invention can be used for various analyzes or diagnoses using hyaluronic acid. Examples of analyzes and diagnoses that can be performed using the hyaluronic acid in sebum prepared according to the present invention include analysis of a subject's skin condition, such as evaluation or future prediction of skin health, and diagnosis of skin diseases. Alternatively, examples include prognosis diagnosis, efficacy evaluation of external skin preparations, prognosis diagnosis, and evaluation of minute changes in the skin.

(Hyaluronic acid detection method)
The method for measuring hyaluronic acid derived from the subject's skin cells prepared by the method of the present invention is not particularly limited and may be any conventionally used method. Examples include immunochromatography and binding protein assay. As a binding protein assay method, a solid phase method, a competitive method, an agglutination method, or a nephelometric method can be used.

The measurement method according to the present invention will be explained below using a sandwich binding protein assay as an example. According to the sandwich binding protein assay method, a buffer containing a surfactant is added to a reaction solid phase, and a sample containing hyaluronic acid is added to the hyaluronic acid-binding protein fixed to the solid phase. The hyaluronic acid-binding protein and the sample hyaluronic acid are combined, and then a hyaluronic acid-binding protein and a labeling substance are added respectively, or a hyaluronic acid-binding protein that has been previously labeled with a labeling substance is added. A sandwich-like bond is formed between the hyaluronic acid-binding protein fixed on a solid phase, the sample hyaluronic acid, and the labeled hyaluronic acid-binding protein, and the labeling substance in the sandwich-like bond causes the sample hyaluronic acid to be Quantify.

Specifically, first, a hyaluronic acid-binding protein is fixed to the surface of a solid phase of a reaction plate. At this time, there is also a surface area on the solid phase to which hyaluronic acid-binding proteins or other molecular species can adhere, and other blood components contained in the sample when adding the sample to be measured. may become stuck. For this reason, it is preferable to add a block before adding the sample to cover the areas to which the hyaluronic acid-binding protein is not attached. Examples of such blocks include γ-globulin, serum albumin, serum, and surfactants such as Tween 20, which can be collected from cows and the like.

Next, a sample containing hyaluronic acid extracted with a surfactant is added to the solid phase to which the hyaluronic acid-binding protein is fixed. In measuring hyaluronic acid, it is known that most of the hyaluronic acid contained in a sample forms proteoglycans in tissues in living organisms, and is bound to proteins, carbohydrates, etc. in serum. Therefore, when performing a binding protein assay, hyaluronic acid cannot be accurately measured unless the binding is dissociated. In the measurement method of the present invention, proteins, sugars, etc. bound to hyaluronic acid can be dissociated by adding a surfactant as a base. After the polymeric hyaluronic acid is bound to the hyaluronic acid-binding protein fixed to the solid phase in this manner, it is preferable to wash the surface of the solid phase.

Next, the labeling substance possessed by the sandwich-like conjugate is measured to quantify hyaluronic acid. At this time, only hyaluronic acid can be quantified by measuring the concentration of the labeling substance on the solid phase surface. The method for measuring the concentration of the labeling substance varies depending on the labeling substance, but for example, when a chemiluminescent substance is used as the labeling substance, the luminescence intensity of the solution after reaction with the substance may be measured. In this case, it is preferable to prepare in advance samples with known concentrations of the labeling substance and hyaluronic acid to be used, and to create a calibration curve regarding the relationship between the signal intensity and the known concentrations.

Examples of the solid phase used in the sandwich binding protein assay method described above include plates, tubes, beads, membranes, gels, latex, etc., but are not particularly limited to these. Any solid phase can be used as long as it is a transparent solid phase. Further, examples of the hyaluronic acid-binding protein used in the sandwich binding protein assay method include proteoglycan, linked protein, hyaluronectin, and the like.

(Hyaluronic acid preparation kit)
In a further aspect, the invention provides a kit for the preparation of hyaluronic acid derived from skin cells of a subject, comprising a device for collecting sebum from the subject. Examples of sebum collecting tools include sebum-absorbing or adhesive materials, instruments for scraping sebum from the skin, and the like. The sebum-absorbing material is preferably a flexible sheet-like material made from a material such as polypropylene. Preferred examples of the sebum-absorbing material include oil blotting paper and oil blotting film. The sebum-absorbing material is preferably in a dry state, containing no water-soluble solvent or water. The sebum-adhesive material is preferably in the form of a sheet or a plate, and if necessary, an adhesive such as poly-L-lysine for adhering sebum may be applied to the surface thereof. Preferred examples of the sebum-adhesive material include glass plates, tapes, and the like. Preferred examples of instruments for scraping off sebum include a spatula, a scraper, and the like.

Preferably, the kit for preparing hyaluronic acid according to the present invention further includes a reagent for separating hyaluronic acid from sebum collected with the collection tool. For example, when the measurement method is a sandwich binding protein assay, basically a solid phase body to which a hyaluronic acid-binding protein is fixed, a buffer containing a surfactant, and a hyaluronic acid-binding protein to be added are used. , and a labeling substance for quantifying sample hyaluronic acid, and these four components can be stored in separate containers and stored as a kit that can be used according to the prescription at the time of use. It is also possible to further add to the kit the above-mentioned kit, such as a block material for covering the portion to which the hyaluronic acid-binding protein is not attached, and a cleaning solution for the solid phase surface that is usually easily available. Furthermore, the hyaluronic acid-binding protein for addition may be labeled in advance with the labeling substance. Furthermore, the portion of the surface of the solid phase to which the hyaluronic acid-binding protein is fixed, to which the hyaluronic acid-binding protein is not fixed, may be covered with a block. The block may be one selected from the group consisting of γ-globulin, serum albumin, serum, and Tween20. The solid phase can be selected from the group consisting of plates, tubes, beads, membranes, gels, and latex, and the labeling substance can be selected from isotopes, fluorescent dyes, avidin, biotin, chemiluminescent substances, and enzymes. be able to.

Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited thereto.
(1) Preparation of hyaluronic acid using sebum as a sample In order to align the collection conditions, the facial sebum of the five subjects was first removed using a blotting film one hour before the actual collection. After one hour, sebum was collected from each subject's face again using the oil-blotting film. Next, the oil-blotting film with facial sebum attached was cut into an appropriate size and placed in a Corning tube. Furthermore, a buffer containing a surfactant (RIPA buffer) was added to the extent that the oil-blotting film was immersed. Then, the Corning tube was vigorously agitated and shaken to extract hyaluronic acid. In addition, as a control, the same preparation operation was performed using an unused oil-blotting film. In addition, the amount of lipid collected was calculated from the weight of the oil-blotted film before and after collection.

(2) Measurement of hyaluronic acid Subsequently, hyaluronic acid was measured using Hyaluronan Duoset ELISA (R&D Systems, Inc.). According to the attached protocol, hyaluronic acid contained in the hyaluronic acid extract prepared earlier was quantified. The results are shown in Figure 1.

As shown in FIG. 1, hyaluronic acid was detected in all subjects. On the other hand, no sebum was detected in the unused oil blotting film. This suggests that sebum contains hyaluronic acid. Furthermore, the amount of hyaluronic acid detected varied by up to four times between subjects. The cause of the variation between subjects is not clear, but it is thought that individual differences may be contributing.

Next, we investigated solvents that can extract hyaluronic acid. The test was conducted using the sebum of Subject E, who had a high amount of hyaluronic acid detected in the previous test. Instead of RIPA buffer, water, organic solvent (ethanol, acetone, hexane, hexane-methanol, acetone-diethyl ether, chloroform-ethanol), surfactant (SDS, Triton X-100, sodium deoxycholate, myristyl sulfobetaine) ) is the same as before except that it is used. Note that the Bligh-Dyer method was also investigated. The results are shown in Figure 2.

As is clear from FIG. 2, hyaluronic acid could not be detected in water or organic solvent. On the other hand, when a surfactant was used, hyaluronic acid could be extracted with any surfactant. Furthermore, among the surfactants used, SDS and sodium deoxycholate were able to extract the same amount as RIPA Buffer, and Triton X-100 and myristyl sulfobetaine were able to extract even more hyaluronic acid than RIPA Buffer. . Furthermore, even with the Bligh-Dyer method, which allows collection of water-soluble fractions, detection was only to the same extent as when extraction was performed with water, suggesting that extraction efficiency would be higher if only a surfactant was used. It was done.

As explained above, according to the present invention, it has become clear that hyaluronic acid can be detected in sebum, which was conventionally thought not to contain hyaluronic acid, by a simple method. This allows hyaluronic acid to be detected non-invasively and directly, making it possible to observe changes in the skin over time.

Claims (5)

A method for preparing hyaluronic acid derived from skin cells of a subject, the method comprising separating hyaluronic acid from skin surface lipids collected from the subject. 2. The preparation method of claim 1, further comprising collecting lipids on the skin surface of the subject. The preparation method according to claim 1 or 2, wherein the hyaluronic acid of the subject is derived from at least a portion of the epidermis, sebaceous glands, hair follicles, and sweat glands. A method for detecting hyaluronic acid derived from skin cells of a subject, the method comprising:
a collection step of collecting lipids on the skin surface of the subject;
a treatment step of treating the collected skin surface lipids with a surfactant;
a detection step of detecting hyaluronic acid treated with a surfactant;
A method for detecting hyaluronic acid derived from skin cells of a subject, comprising: 5. The detection method according to claim 4, wherein the surfactant is one of anionic, amphoteric, or nonionic.

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